The immune system has evolved as a complex network of mechanisms to discriminate between ‘self and non-self,’ and homeostasis is reached by a tight control that leads to recognition and elimination of foreign antigens and/or development of tolerance. T-lymphocytes are one of the main characters of cellular immunity, as maintaining the balance between pro-inflammatory (Th1/Th17 cells) and anti-inflammatory (Th2/Treg) populations is essential for resolution of inflammation, keeping autoimmune and chronic inflammatory diseases at bay.
Amongst the different regulatory circuits that shape this equilibrium (immune homeostasis) are cell surface glycosylation and lectin-glycan signaling. Lectins are proteins with affinity for carbohydrates that induce particular cascade responses, and thus modulate the immune response. This regulation appears to be context dependent, namely: on the glycan side, different outcomes are achieved by programmed remodeling of the cell-surface glycome through the sequential actions of glycosidases and glycosyltransferases; and on the lectin side, microenvironmental conditions can alter lectin affinity and binding capability.
Galectins are members of a family of multifunctional lectins that are defined by their specificity for β-galactoside-containing glycans and a carbohydrate recognition domain (CRD). Cooper, D. N. W., “GALECTINOMICS: FINDING THEMES IN COMPLEXITY,” Biochimica et Byiophysica Acta, General Subjects, 1572:209-231 (2002). In humans, CRDs have been identified for approximately 16 different galectins, a central example being Galectin-1 (Gal-1), a lectin that specifically binds N-acetyllactosamine terminal moieties exposed on cell surfaces and cross-links to a preferred set of glycosylated receptors to transduce signals that directly lead to Th1 and Th17 apoptosis and termination of the inflammatory response. Human Gal-1 is a small lectin composed of 135 amino acids, which folds into a three-dimensional structure in the form of a β-sandwich of two slightly bent sheets with variable long connecting loops. A notable feature of Gal-1 is the high proportion of cysteine residues (Pe'er et al., “PROTEOMIC SIGNATURES: AMINO ACID AND OLIGOPEPTIDE COMPOSITIONS DIFFERENTIATE AMONG PHYLA,” Proteins, 54:20-40 (2004)), each Gal-1 monomer containing six cysteines: Cys2, Cys16, Cys60, Cys88, and Cys130.
Binding of Gal-1 depends on glycosyltransferase activity, including the activity of N-acetylglucosaminyltransferase 5 (GnT5), an enzyme responsible of generating β-1,6-N-glycan branch structures and a core 2 β-1,6 N-acetylglucosaminyltransferase (GCNT1) that elongates the core 2-O-glycans. Whereas Th1 cells and Th17 cells express the repertoire of cell surface glycans that are critical for Gal-1 binding and cell death, Th2 cells are protected from Gal-1 binding through α-2,6 sialylation of cell surface glycoproteins (Toscano et al., “DIFFERENTIAL GLYCOSYLATION OF TH1, TH2 AND TH-17 EFFECTOR CELLS SELECTIVELY REGULATES SUSCEPTIBILITY TO CELL DEATH,” Nat. Immunol., 8:825-34 (2007)), a modification that involves α(2,6) sialyltransferase (ST6) and thereby prevents Gal-1 binding by masking galactose residues on LacNAc units. The anti-inflammatory activity of Gal-1 is not limited to T-cell apoptosis; it has also been found to promote differentiation of tolerogenic dendritic cells (Ilarregui et al., “TOLEROGENIC SIGNALS DELIVERED BY DENDRITIC CELLS TO T CELLS THROUGH A GALECTIN-1-DRIVEN IMMUNOREGULATORY CIRCUIT INVOLVING INTERLEUKIN 27 AND INTERLEUKIN 10,” Nat. Immunol., 10:981-991 (2009)), and to favor conversion of macrophages toward a M2-type phenotype (Starossom et al., “GAL-1 DEACTIVATES CLASSICALLY ACTIVATED MICROGLIA AND PROTECTS FROM INFLAMMATION-INDUCED NEURODEGENERATION,” Immunity, 37(2):249-63 (2002)). In fact, administration of recombinant Gal-1 has been found to ameliorate disease severity in several autoimmune models of arthritis, uveitis, and TNBS-induced colitis. See Toscano et al., Journal of Immunology, 176:6323-32 (2006); and Santucci et al., “GALECTIN-1 SUPPRESSES EXPERIMENTAL COLITIS IN MICE,” Gastroenterology, 124 (5): 1381-94 (2003).
The therapeutic potential of Gal-1 is, however, limited by intrinsic biochemical factors, including its sensitivity to oxidation and acidic pH, both of which are conditions typically involved in inflammatory microenvironments. Moreover, as most studies to date regarding Gal-1 function have been performed at normal physiological conditions (i.e., a pH of about 7.4), most of the available physicochemical data characterizing activity and affinity of Gal-1 does not reflect its role in an inflammatory locus where extracellular acidosis can make the pH fall below 5.5. This high proton concentration is normally attributed to infiltration and activation of inflammatory cells, leading to increased oxygen demand and energy, accelerated glycolysis, and increased lactic acid secretion. Menkin, Science (1956). Furthermore, although lactic acid (i.e., extracellular acidosis) has been shown to influence many processes related to the immune metabolism ((Geffner et al., (1993); Jancic et al., (2012); Kraus & Wolf, (1996); Martinez et al., (2007); Trevani et al., (1999); Vermeulen et al., (2004)), little is known about the mechanisms by which cell communication is influenced by these conditions.
It is therefore an object of the invention to investigate the effect of altered extracellular pH, particularly that of an acidic microenvironment, on immune cells and their function. More specifically, it is an object of the invention to investigate how Gal-1 affects immune cells and their function.